U.S. patent number 4,882,423 [Application Number 07/032,025] was granted by the patent office on 1989-11-21 for substance-conjugated complement component c1q.
This patent grant is currently assigned to Calpis Food Industry, Fumiaki Taguchi. Invention is credited to Kunio Ezawa, Kenichi Fukunaga, Kinichi Hara, Masaro Hayashi, Jun Kuranari, Isamu Mitsui, Fumiaki Taguchi.
United States Patent |
4,882,423 |
Taguchi , et al. |
November 21, 1989 |
Substance-conjugated complement component C1q
Abstract
A substance-conjugated complement component Clq is provided. A
substance such as signal emitting substances or cell function
regulating substances is conjugated via a sulfur atom to at least
one site of the component. The site is not involved in binding
immunoglobulins. A marker-labelled complement component Clq is used
for measuring a complement-binding antibody, an antigen, a
neutralizing antibody or a substance produced internally of and at
the surface of a cell or a microorganism by measuring the
marker.
Inventors: |
Taguchi; Fumiaki
(Sagamihara-shi, Kanagawa-ken, JP), Mitsui; Isamu
(Yokohama, JP), Hara; Kinichi (Yokohama,
JP), Hayashi; Masaro (Yokohama, JP), Ezawa;
Kunio (Tokyo, JP), Fukunaga; Kenichi (Tokyo,
JP), Kuranari; Jun (Tokyo, JP) |
Assignee: |
Calpis Food Industry (both of,
JP)
Taguchi; Fumiaki (both of, JP)
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Family
ID: |
27572627 |
Appl.
No.: |
07/032,025 |
Filed: |
March 30, 1987 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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779671 |
Sep 24, 1985 |
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Foreign Application Priority Data
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Oct 2, 1984 [JP] |
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59-205686 |
Oct 25, 1984 [JP] |
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59-223049 |
May 17, 1985 [JP] |
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60-103898 |
Jul 24, 1985 [JP] |
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60-162012 |
Jul 29, 1985 [JP] |
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60-166004 |
Mar 31, 1986 [JP] |
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61-70936 |
Mar 31, 1986 [JP] |
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61-70937 |
Mar 31, 1986 [JP] |
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61-70938 |
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Current U.S.
Class: |
530/380;
514/19.3; 514/7.6; 514/9.7; 514/2.4; 435/7.9; 435/4; 435/7.2;
435/7.22; 435/7.32; 435/7.7; 435/14; 435/21; 435/28; 530/350;
530/408; 435/7.21; 435/7.31; 435/7.4; 435/7.72; 435/7.92; 435/26;
530/404 |
Current CPC
Class: |
A61K
49/001 (20130101); A61K 49/0043 (20130101); A61K
49/0056 (20130101); A61K 51/04 (20130101); A61K
51/081 (20130101); G01N 33/564 (20130101); G01N
33/58 (20130101) |
Current International
Class: |
A61K
49/00 (20060101); A61K 51/04 (20060101); A61K
51/02 (20060101); A61K 51/08 (20060101); G01N
33/58 (20060101); G01N 33/564 (20060101); C07K
015/00 () |
Field of
Search: |
;530/404,408,350,380
;435/4,7,14,21,26,28 ;514/2,3,6 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Smith et al-J. of Immunol. Methods vol. 67 (1984) pp. 167-172.
.
Alcolea et al.-Mol. Immunol. vol. 23(1) (1986) pp. 39-44. .
Alcolea et al.-Chem. Abst. vol. 104 (1986) p. 86773e. .
Reid et al.--Chem. Abst. vol. 101 (1984) p. 53053e. .
Nishioka et al.--Chem. Abst. vol. 97 (1982) p. 196,644h. .
Bing et al.--Chem. Abst. vol. 97 (1982) p. 53755j. .
Ingham et al.--Chem. Abst. vol. 98 (1983) p. 177,328n..
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Primary Examiner: Rosen; Sam
Attorney, Agent or Firm: Darby & Darby
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part application of
application Ser. No. 779,671 filed Sept. 24, 1985, now abandoned.
Claims
What is claimed is:
1. A complement component C1q wherein a substance is conjugated via
a sulfur atom to at least one site of said component, said site not
being involved in binding immunoglobulins,
said substance being selected from the group consisting of signal
emitting substances and cell function regulating substances, said
signal emitting substance being a member selected from the group
consisting of enzymes, coenzymes, enzyme substrates, dyestuffs and
pigments, magnetizable substances, donors and acceptors for
electron transference, radioactive substances, metal compounds,
metal compositions and agglutinating substances,
said cell function regulating substance being selected from the
group consisting of antibiotics, growth factors, hormones, cell
activation factors, cell division factors, anticancer agents and
toxins.
2. The complement component C1q according to claim 1, wherein said
enzyme is selected from the group consisting of peroxidases,
alkaline phosphatases, galactosidases and alcohol
dehydrogenases.
3. The complement component C1q according to claim 1, wherein said
coenzyme is selected from the group consisting of nicotinamide
adenine dinucleotide, nicotinamide adenine dinucleotide phosphate,
flavin adenine dinucleotide and flavin adenine dinucleotide
phosphate.
4. The complement component C1q according to claim 1, wherein said
enzyme substrate is selected from the group consisting of
o-nitrophenyl-.beta.-D-galactopyranoside and 3-hydroxysteroid.
5. The complement component C1q according to claim 1, wherein said
dyestuff and pigments are selected from the group consisting of
hemoglobin, methylene blue and fluorescein isothiocyanate.
6. The complement component C1q according to claim 1, wherein said
magnetizable susbstance is selected from the group consisting of
carbonic iron, iron-containing microcapsules and complexes of iron
with proteins.
7. The complement component C1q according to claim 1, wherein said
donor and acceptor for electron transference are chlorophyl.
8. The complement component C1q according to claim 1, wherein said
radioactive substance is selected from the group consisting of
.sup.124 I-labelled albumin, p-chloro(.sup.203 Hg)mercuribenzoic
acid, N-ethyl(2,3-.sup.14 C)maleimide and iode(1-.sup.14
C)acetamide.
9. The complement component C1q according to claim 1, wherein said
metal compound and said metal composition are selected from the
group consisting of gold colloid and iron-containing
microbeads.
10. The complement component C1q according to claim 1, wherein said
antibiotic is selected from the group consisting of amphotericin B
and actinomycin D.
11. The complement component C1q according to claim 1, wherein said
growth factor is selected from the group consisting of selenium
compounds, insulin, transferrin and epidermal growth factor.
12. The complement component C1q according to claim 1, wherein said
hormone is corticosteroid.
13. The complement component C1q according to claim 1, wherein said
cell activation factor is a macrophage activation factor.
14. The complement component C1q according to claim 1, wherein said
cell division factor is a B cell division factor.
15. The complement component C1q according to claim 1, wherein said
anticancer agent is mitomycin C.
16. The complement component C1q according to claim 1, wherein said
toxin is ricin of toxin of castor bean.
17. The complement component C1q according to claim 1, wherein said
agglutinating substance is selected from the group consisting of
fine particles of latexes, fine particles of microbeads, a
complement component C1q, a combination of an antibody and an
antigen corresponding to said antibody, a combination of lectin and
a material having saccharide structure corresponding to said
lectin, a combination of biotin and avidin, and a combination of
protein and IgM.
18. A process for preparing a substance-conjugated complement
component C1q, comprising the steps of:
(a) adding a reducing agent to a complement component C1q to cleave
at least one S--S bond present at a site not involved in binding
immunoglobulins thereby to obtain a reduced complement component
C1q having at least one exposed --SH group; and
(b) conjugating a substance to said complement component C1q via
said exposed --SH group.
19. The process according to claim 18, wherein said reducing agent
is a sulfur-containing compound.
20. The process according to claim 19, wherein said reducing agent
is selected from the group consisting of mercaptoethylamine,
dithiothreitol, 2-mercaptoethanol, cysteine and glutathione.
21. The process according to claim 18, wherein said reducing agent
is allowed to act on the complement component C1q which is
dissolved in a buffer solution for permitting the complement
component C1q to be present stably.
22. The process according to claim 18, wherein said buffer solution
is selected from the group consisting of tris buffered saline and
phosphate buffered saline.
23. The process according to claim 18, wherein said reducing agent
is allowed to act on said complement component C1q at -2.degree. C.
to 45.degree. C. for 30 seconds to 24 hours.
24. The process according to claim 18, further comprising a step of
removing excess reducing agent after the completion of said step
(a).
25. The process according to claim 18, wherein said substance is
selected from the group consisting of signal emitting substances
and cell function regulating substances.
26. The process according to claim 18, wherein said substance is
conjugated directly to said exposed --SH group.
27. The process according to claim 18, wherein said substance is
conjugated to said exposed --SH group indirectly via a second
susbstance having coupling function.
28. The process according to claim 18, wherein said second
substance having said coupling function has a group selected from
the group consisting of maleimide residue and --SH group, and also
has a group for coupling said substance.
29. The process according to claim 18, wherein said substance is
peroxidase extracted from horseradish, and wherein said second
substance having said coupling function is N-hydroxysuccinimide
ester of maleimide.
30. The process according to claim 18, wherein said substance is
conjugated to said reduced complement component C1q in a buffer
solution for both of said substance to be conjugated and said
reduced complement component C1q.
31. The process according to claim 18 wherein said step (b) is a
step of conjugating said substance indirectly via a second
susbstance having coupling function and via said exposed --SH group
to said complement component C1q, and wherein said step (b) is
effected in a common buffer for said reduced complement component
C1q, said second substance having coupling function and said
substance to be conjugated.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention;
This invention relates to a substance-conjugated complement
component C1q and a process for preparing the same, and a method
for measurement, using the substance-conjugated complement
component C1q. More particularly, it relates to a complement
component C1q conjugated with various substances including markers
and cell function regulating substances and a process for preparing
the same, and also to a process for measurement, using such a
substance-conjugated complement component C1q.
2. Related Art Statement;
It has hitherto been known to utilize the complement fixation
reaction for the measurement or determination of antibodies in
blood serum and antigens, such as microorganisms, phisiologically
active substances and chemicals. This known method makes use of the
serial reactions wherein complement components C1 to C9 are bound
successively to an antibody specifically bound to an antigen. In
detail, this known method comprises the step of adding an excess
amount of complement components to the formed antigen-antibody
complex, the step of determining an amount of residual complement
components through the hemolysis, and the step of determining the
amount of fixed complement components from the degree of hemolysis.
The quantity of the antigen or antibody is then estimated from the
results of the amount of the fixed complement components. In the
hemolysis, complement components act on the sensitized erythrocytes
including sheep red blood cells and anti-sheep red blood cell
antisera so that the complement components may be determined while
using the hemolysis of the sheep red blood cells as the index.
However, practical determination operation of the hemolysis is
extremely complex and needs high level skill and knowledge. In
addition, this known method has a relatively low sensitivity and
requires two days for the determination operation.
Various methods have been proposed to overcome the aforementioned
disadvantages of the known method as described in the preceding
paragraph. For example, Japanese Patent Laid-Open Publication No.
43498/1980 discloses one of such methods. In the method proposed by
the antecedent Publication referred to above, an antibody which
binds, as an antigen, a complement component being bound to another
antibody is labelled with an enzyme, and the amount of the thus
labelled antibody is determined by the enzymatic activity thereof.
This method is, therefore, one of the so-called enzyme-labelled
antibody techniques. However, this method involves two step
reactions, since a labelled antibody which binds, as an antigen, a
complement component must be used. Accordingly, rinsing operations
are required after each of the reactions, leading to increase in
labor and time. In fact, this determination method costs much time
as several hours.
On the other hand, a method of determining a neutralizing antibody
has been made known, for example, by Takashi Kitamura, "Tissue
Culture Technology for Inspection of Virus", published by KINDAI
SHUPPAN (1980), page 246. When an antibody against poliovirus, for
instance, is determined by this method, cultivated cells originated
from human being, a monkey or an ape are first inoculated with the
poliovirus. (Meanwhile, the poliovirus does never grow if it is
inoculated into cells originated from the sources other than human
being, a monkey or an ape.) The cells inoculated with the
poliovirus collapse and are deseased as the result of
cytopathogenesis due to propagation of the virus. However, the
reaction product of a neutralizing antibody and the virus, (the
infectiousness of virus being neutralized by the neutralizing
antibody), can not propagate even if it is inoculated upon a cell
originated from human being or monkey so that the cell is kept to
have normal form and functions. Making use of this principle, a
specific virus is reacted with blood serum and then the titre of
the neutralizing antibody is determined by inspecting the presence
or absence, and the degree if present, of plaque and CPE
(cytopathogenic effect).
However, when the poliovirus is determined by the method described
in the preceding paragraph, the poliovirus must be cultivated for
about 7 days in a normal test in addition to the fact that the
inspection and judgement of the result should be made by a skilled
person rather than being easily conducted by a person having
ordinary or middle level skill. For this reason, an order of test
for the determination of neutralizing antibody is not accepted even
by a large scale inspection center at the present day.
On the other hand, as a method for determining antigens or
antibodies in a simpler way, there has been known in the art a
method wherein properties of complement component C1q binding an
antigen-antibody complex is utilized. (Simpson et al.,"Jounal of
Immunological Methods", Vol. 67, 167 to 172 (1984).) In this known
method, glutalaldehyde or periodic acid is conjugated to the
complement component C1q as a cross-linker, and peroxidase (enzyme)
is conjugated via said cross-linker to the complement component C1q
as a marker. Marchalonis J. J., "Biochemical Journal", Vol. 113,
pp229 to 305 (1969) discloses a method in which radioactive iodine
is conjugated to the complement component C1q through the
chloramine T method as a marker. However, in these known methods,
an enzyme or radioactive iodine is coupled with each of the
complement component C1q molecules via an amino group present on
the molecule generally and at random, resulting in entire
modification of the molecule since the very site of each molecule
having inherent properties capable of binding to an immunocomplex
has been chemically modified by said cross-linker or coupler.
Accordingly, the binding activity of such a marker-labelled
complement component C1q for binding to an antigen-antibody complex
is seriously lowered to a level not to adapt for quantitative
measurement as a reagent. Moreover, a false-positive reaction takes
place frequently by the latent presence of said cross-linker in the
marker-labelled complement component C1q to make it impossible to
continue the determination operations. It has, thus, been
impossible to provide a reliable determination method for
determining an antigen or antibody in a precise and reproducible
manner by the use of the complement component C1q.
OBJECTS AND SUMMARY OF THE INVENTION
It is, therefore, a primary object of this invention to provide a
complement component C1q which is conjugated with a variety of
substances, such as markers or cell function regulating substances,
while preserving its inherent binding capacity for binding with an
immunocomplex without any detraction, and a process for preparing
such a complement component C1q, and to provide a determination
method in which such a complement component C1q is used in a
substance-conjugated condition.
Another object of this invention is to provide a complement
component C1q which is conjugated with a variety of substances to
be conveniently used as a determination or detection reagent for
determining or detecting a specific antigen present in a body fluid
or held or bound to a cell or body tissue or for determining or
detecting the corresponding antibody for the specific antigen, a
modified immunoglobulin or immunocomplex, and a process for
preparing such a complement component C1q, and to provide a
determination method in which such a complement component C1q is
used in a substance-conjugated condition.
A further object of this invention is to provide a complement
component C1q which is conjugated with a variety of substances to
be conveniently used as a curing agent or medicine for regulating
physiological function of a variety of cells which have cell
surface structures identified by specific antibodies or which can
capture specific immunocomplexes or complements, and a process for
preparing such a complement component C1q, and to provide a
determination method in which such a complement component C1q is
used in a substance-conjugated condition.
A still further object of this invention is to provide a
determination method utilizing a complement component C1q, which is
simple and easy in determination operation and can be conducted for
a short operation time even by an unskilled person.
Yet a further object of this invention is to provide a
determination method utilizing a complement component C1q, which
has high sensitivity to give reproducible result of
determination.
The above and other objects of this invention will be apparent from
the following detailed description thereof.
According to the present invention, there is provided a complement
component C1q wherein a substance is conjugated via a sulfur atom
to at least one site of said component, said site being not
involved in binding immunoglobulins.
Also provided in accordance with this invention is a process for
preparing a substance-conjugated complement component C1q,
comprising the steps of:
(a) adding a reducing agent to a complement component C1q to cleave
at least one S--S bond present at a site not involved in binding
immunoglobulins thereby to obtain a reduced complement component
C1q having at least one exposed --SH group; and
(b) conjugating a substance to said complement component C1q via
said exposed --SH group.
Further provided in accordance with the invention is a method for
measurement by the use of a complement component C1q comprising
reacting a maker-labelled complement component C1q with a material
to be measured, said complement component C1q being conjugated with
the marker at at least one site not involved in binding
immunoglobulins, thereby to obtain a reaction material having said
marker, and measuring said marker.
BRIEF DESCRIPTION OF THE DRAWING
The single FIGURE, FIG. 1, of the appended drawing is a graph
showing the change in neutralizing antibody titer of an anti-HSV
positive human serum in Example 15, one of the examples of the
invention.
DESCRIPTION OF THE INVENTION
The present invention will now be decribed in detail.
In general, after an antigen is bound to an antibody, a complement
is bound to the antibody already bound with the antigen to destruct
the antigen. The complement includes complement components C1q,
C1s, C1r, C2a, C2b, C3a, C3b, C4b, C5b, C6, C7, C8 and C9, and each
one of these complement components binds to a specific antibody
pertinently depending on the specific immunological reaction or
allergy rection. These complement components bind in a fixed order
such that the component C1q binds to the antigen at a first place,
followed by binding of C1s and C1r to C1q, and then the other
complement components bind serially.
After eager investigations with the estimation that a
substance-conjugated complement component having a utility when
used as a determination reagent or a curing agent might be prepared
by conjugating a variety of substances to the complement component
C1q, which is the component coupled with an antigen-antibody
complex at the first place, such that the subsequent binding
between the immunoglobulins acting as an antibody and the component
C1q is not hindered, we have succeeded to allow a variety of
substances to conjugate at sites of the complement component C1q
other than the sites at which the immunoglobulins are to be
bound.
In detail, we have given attention to the presence of nine S--S
bonds located at the sites of the polypeptide molecule of the
complement component C1q, the S--S bonds locating at the sites
remote enough for affecting the site having the binding capability
for the immunoglobulins and being formed by the fourth cysteine
residues from the N-terminals of the A, B and C chains of the
polypeptide constituting the complement component C1q so that they
are apt to be attaked by a reducing agent and apt to conjugate with
a substance readily.
In view of the presence of such S--S bonds, we have contemplated to
cleave these S--S bonds by the action of a reducing agent to expose
at leat one S--H group in a step (a) of the process provided by the
present invention.
The reducing agents which may be conveniently used in this step (a)
include those used conventionally, the examples being
sulfur-containing compounds, such as mercaptoethylamine,
dithiothreitol, 2-mercaptoethanol, cysteine and glutathione.
The reducing step is carried out under the condition that the
complement component C1q is not modified. Preferably, reducing may
be effected by dissolving the complement component C1q in a buffer
solution in which it exists stably and then it is attacked by a
reducing agent. Examples of the buffer solution used for this
purpose include a tris buffered saline containing 10% of sucrose, 1
mol of sodium chloride and 5 mM (millimols) of sodium
ethylenediamine tetra-acetate, and a phosphate buffered saline
(PBS). The reducing reaction is carried out, generally, at about
-2.degree. C. to 45.degree. C. for about 30 seconds to 24 hours,
the reaction temperature and time being changed depending on the
specific reducing agent used.
It is desirous that the thus reduced complement component C1q be
stored in a buffer solution to be used in the subsequent step (b)
after removing the excess reducing agent by means of a conventional
method, such as dialysis, salting-out process or gel
filtration.
The complement component C1q utilized in the present invention is a
glycoprotein contained in the blood serum of animal, and has nature
for binding firmly to the immunoglobulins when the immunoglobulins
contained similarly in the blood serum and acting as an antibody
react specifically with the corresponding antigen. The complement
component C1q used in the invention may be isolated from various
aminals including sheep, rabbit, guinea pig, cattle, horse, dog,
mouse and human being, and the fraction enriched in the C1q
component may be picked up in accordance with a conventional
purifying operation. (In this connection, reference should be made
to "Operations in Immunological Experiment B", published by the
Japanese Immunological Society, pp 1376 to 1380 (1974), if
necessary.)
In the process of the invention, a variety of substances is
conjugated via the exposed SH group of the reduced complement
component C1q at the subsequent step (b). The substances to be used
in the step (b) and to be conjugated with the complement component
C1q via the exposed SH group include signal emitting substances,
such as enzyme substrates, dyestuffs or pigments, magnetizable
substances, donors or acceptors for electron transference,
radioactive materials, metal compounds and metal compositions,
which emit signals detectable by the sensory organs or external
instruments, enzymes or coenzymes which may be modified to emit
detectable signals or agglutinating substances which get together,
thereby to be readily detected; cell function regulating
substances, for example, certain enzymes which act on the
counter-substances conjugated to the complement component C1q to
provide the latter with any functions; and substances, such as high
polymer materials, which capture or fix the counter-substances
conjugated to the complement component C1q.
More specifically, examples of the enzyme substrate are
o-nitrophenyl-.beta.-D-galactopyranoside and 3-hydroxysteroid; and
dye stuffs or pigments include hemoglobin, the redox dyestuffs,
such as methylene Blue, and fluorescent dyestuffs, such as
fluorescein isothiocyanate. Examples of the magnetizable substance
are organic irons, such s carbonic iron, and microcapsules
containing iron, and complexes of iron with proteins may also be
used. The donors and acceptors for electron transference include a
wide variety of substances which take part in the electron
transference, and chlorophyll which may be energized to take part
in the electron transference is included in this group of substance
and preferably used in the invention. Examples of the radioactive
substances are .sup.124 I-labelled albumin, p-chloro(.sup.203
Hg)mercuribenzoic acid, N-ethyl(2,3-.sup.14 C)maleimide and
iode(1-.sup.14 C)acetamide.
The metal compounds and compositions, other than the corbonic iron
referred to hereinabove, which may be used in the invention include
gold colloid and iron-containing microbeads.
Examples of the enzymes are peroxidase, alkaline phosphatase,
galactosidase and alcohol dehydrogenase; wheras examples of the
coenzymes are nicotinamide adenine dinucleotide, nicotinamide
adenine dinucleotide phosphate, flavin adenine dinucleotide and
flavin adenine dinucleotide phosphate.
Example of the agglutinating substances are agglutinative fine
particles such as latexes or microbeads; and other complement
components C1q, i.e. complement components C1q to be conjugated
with the exposed SH group of the reduced complement component C1q.
These substances per se form agglutinate masses, which may be
conjugated with the reduced complement component C1q. The other
examples of the agglutinating substances include a combination of
antibodies such as IgG or IgM and antigens corresponding to the
respective antibodies, a combination of lectins such as peanut
lectin or wheat embryo lectin and materials having the
corresponding saccharide structures, a combination of biotin and
avidin and a combination of protein and IgM. In a case where these
combinations are used, one material forming the combination is
conjugated with the reduced complement component C1q and then the
other material forming the combination is added to form agglutinate
masses. If the agglutinating substances are used as a marker, the
marker is easily detected by turbidity.
A variety of substances may be included in the cell function
regulating substance and conveniently used in the invention,
examples being surface active agents; antibiotics having activities
to vital membranes, such as amphotericin B, and antibiotics
affecting the metabolisms of cells, such as actinomycin D; trace
essential nutrients or growth factors indispensable for the growth
of cells, such as selenium compounds, insulin, transferring and
epidermal growth factor; hormones such as corticosteroid; factors,
participating in the manifestation of cell function, such as
macrophage activation factor; factors participating in the cell
division, such as B cell division factor; anticancer agents, scuh
as mitomycin C; and toxins such as ricin of toxin of castor
bean.
For instance, an enzyme may be utilized as the signal emitting
substance while being conjugated to the complement component C1q to
act as a marker so that it is used as a determination or detection
reagent for determining or detecting a specific antigen present in
a body fluid or held by or adhering to a cell or body tissue, or
for determining or detecting the corresponding antibody to said
antigen, modified immunoglobulin or an immunocomplex.
When an antibiotic is used as the cell function regulating
substance while being conjugated to the complement component C1q,
the conjugated product may be used as a curing agent for
controlling the physiological function of a cell having a cell
surface structure which can be identified by a specific antibody or
a cell capable of capturing an immunocomplex or a complement.
In the aforementioned step (b) of the process of the invention, a
substance having a group which can be conjugated to the exposed -SH
group may be directly conjugated to the reduced complement
component C1q, or a substance may be conjugated indirectly to the
complement component C1q via a second substance having a coupling
function, for example, via one or more spacers or cross-linkers.
Examples of the substance which may be directly conjugated include
activated thiol Sepharose (Trade Name of Pharmacia Fine Chemicals
Co.) and p-chloromercuribenzoate. Any substance having a group
capable of conjugating to the exposed --SH group of the complement
component C1q, such as maleimide residue or --SH group, and also
having another group capable of coupling with the substance to be
coupled indirectly with the complement component C1q may be used as
the coupling agent for such purpose.
The coupling agent or the second substance having a coupling
function, of course, varies depending on the substance to be
coupled thereby. As an illustrative example, when peroxidase
extracted from horseradich is coupled by the N-hydroxysuccineimide
ester of N-(4-carboxycyclohexylmethyl)maleimide, both reagents are
dissolved in a buffer having a pH value of 6.5 to 7.5 and
containing sodium ethylenediamine tetra-acetate to allow them to
react with eath other at 30.degree. C. for an hour. The reaction
conditions are selected in consideration of the characteristics of
a cross-linker used. Other than the N-hydroxysuccineimide ester of
N-(4-carboxycyclohexylmethyl)maleimide referred to above,
N-hydroxysuccineimide esters of m-maleimide benzoic acid,
N-(4-carboxyphenylmethyl)maleimide and maleimide acetic acid may be
used as the cross-linker. A peroxidase having therein a maleimide
group is produced. The thus produced peroxidase having a maleimide
group is mixed with the reduced complement component C1q, and
maintained in a buffer held at pH 5.5 to 6.5 and containing sodium
ethylenediamine tetra-acetate at 4.degree. C. for 2 hours, whereby
a complement component C1q conjugated with peroxidase is
obtained.
At the final step, the fraction of the complement component C1q
labelled with peroxidase, which serves as a marker, and having the
activities originated both from the peroxidase and the complement
component C1q may be picked up through the gel filtration
As another illustrative example, if an antibody is employed as the
agglutinating substance, an antibody having a maleimide group is
obtained by the similar procedures under the similar conditions as
explained hereinabove with respect to peroxidase extracted from
horseradish. When the antibody having the maleimide group is mixed
and reacted with the reduced complement component C1q, a complement
component C1q conjugated with the antibody is obtained.
As a further illustrative example, if another complement component
C1q is employed as the agglutinating substance, the complement
component C1q is firstly reduced by the procedures described
hereinabove with respect to the step (a). Then, the reduced
complement component C1q is put in a buffer solution containing
ethylenediamine tetre-acetate and N,N'-para-phenylene dimaleimide
at pH 5.5 to 6.5 and is maintained for 20 hours at 4.degree. C., so
that conjugated complement components C1q each conjugated via S--S
bond to form an agglutinate mass and having maleimide groups are
obtained. When the conjugated complement components C1q are mixed
and reacted with the reduced complement component C1q, a complement
component C1q further conjugated with complement components C1q is
obtained.
It is preferred that the step (b) of the process of the invention
be carried out in the presence of a buffer for both of the reduced
complement component C1q and the substance to be conjugated
thereto, and carried out in the presence of a buffer for the
coupler or cross-linker in case where such a substance having the
coupling or cross-linking function is used.
In the substance-conjugated complement component C1q provided
according to this invention, the substance is conjugated at a site
or sites having no binding capacity with the immunoglobulins, so
that the binding capability to the immunoglobulins inherent to the
complement component C1q is kept intact without being hindered by
the conjugating substance.
Since the substance-conjugated complement components C1q provided
according to this invention are conjugated with various signal
emitting substances and cell function regulating substances without
blocking the sites at which the immunoglobulins is to be bound,
they may be used for various applications including determination
or measurement reagents giving the reproducible results or curing
medicines in which their inherent capacities for binding with the
immunoglobulins are utilized.
The method for the measurement or determination, according to the
invention, utilizing the substance-conjugated complement component
C1q will now be described. The signal emitting substances, as
described hereinbefore, may be used in the measurement method
according to the invention. Such a signal emitting substance may be
utilized as a marker. In detail, a substance-conjugated complement
component C1q having a marker conjugated at a site that is not
adapted to bind with the immunoglobulins is prepared, and then the
thus prepared marker-labelled complement component C1q is allowed
to react with another material to be measured, whereby a reaction
product conjugated with the marker is obtained, Subsequently, the
labelled marker is qualitatively or quantitatively analysed to
measure a variety of antigens, antibodies, neutralizing antibodies,
substances produced or appearing in cells or on the surfaces of
cells or microorganisms. In this manner, the method of the
invention may be applied for comprehensive uses, including various
clinical inspections and diagnoses of diseases.
One group or category of the substances which may be measured by
the method of the invention includes complement-binding antibodies.
By the determination of certain complement-binding antibodies,
various diseases of wide-ranging hosts including not only human
beings but also animals and plants may be diagnosed and judgement
may be made whether the hosts are infected or not, the diseases
which may be determined by the method of the invention including
infectious diseases caused by microorganisms, such as bacterium,
chlamydia and virus, tumor and autoimmune diseases such as systemic
lupus erythematodus. Particularly in clinical inspection of such an
infectious disease and autoimmune disease, it is a common practice
to inspect whether or not a specific antibody uniquely appearing
with a certain disease is present in the blood serum. Various
measurement or inspection methods are known, and the
complement-binding reaction is involed in one of the important
inspection methods. Notwithstanding that this complement-binding
reaction has a utility when utilized in a method of inspecting a
certain specific antibody, the reaction has not been frequently
uesd, irrespective of the importance thereof, since it involves
cumbersome determination operations and requires extremely high
level skill.
Under these circumstances, a considerable contribution in
industrial and medical fields is made by the provision of a simple
and speedy method based on this principle is developed to give a
reproducible measurement or determination value at high
sensitivity.
In determination of a complement-binding antibody in accordance
with the present invention, an antigen is initially fixed to a
solid phase or carrier, and then the fixed antigen is allowed to
react with an antibody and with a complement component C1q combined
with a marker, followed by removal of unreacted materials and then
the marker is quantitatively analysed. Utilizable antigens include
viruses and bacteria, such as varicella-zoster virus, measles
virus, rubella virus, influenza virus, herpes simplex virus,
hepatitis virus, mumps virus and mycoplasma phneumonitis;
physiologically active substances such as interferon; and antigens
against autoantibodies such as DNA. Initially, such an antigen is
fixed to a solid carrier to form a solid phase. Whereupon, the
operations, particularly rinsing operations, can be carried out
easily as compared with the prior art technology in which a lquid
phase must be handled. Any carries may be used as far as the
antigen absorbed thereby is not easily released from the solid
phase, the examples being synthetic high polymers such as
polyvinylchloride and polystyrene, natural high polymers such as
filter paper, and cells and tissues. More specifically, a
microtiter plate and polystyrene beads may be referred to as
illustrative examples. The antigen may be fixed to the solid phase
by fixing the same on the surface of the solid carrier through
physical absorption of chemical covalent bond and the like. In case
where a cell or tissue is used as a solid carrier, the antigen may
also be fixed thereto by infection.
Then, the fixed antigen is allowed to react with the specific or
corresponding antibody and a complement component C1q combined with
a marker. The antibody is the objective substance which is to be
measured or inspected by the method of the invention, according to
the aspect of the invention discussed just now, and the examples
thereof are body fluids, such as blood serum, cerebrospinal fluid
and saliva. The blood serum is used most frequently, since it
contains the largest quantities of antibodies. The antibodies
determined or measured by the method of the invention must bind to
the corresponding antigens uniquely or specifically, and must be
capable of binding to the complement components. However, almost
all of the antibodies produced in living bodies satisfy the
aforementioned conditions, and hence they may be measured by the
method of the invention.
The reaction of the antigen fixed to the solid phase, the antibody
and the complement component C1q conjugated with a marker takes
place spontaneously by simply mixing the aforementioned three
reactants together to complete the reaction for forming a combined
product. While the reaction temperature and reaction time vary
depending on the specific antigen or other reactants used, these
conditions may be selected properly unless bioactivities are
lost.
Since the complex composed of the antigen, the antibody and the
marker-labelled complement component C1q is fixed to a solid phase,
the unabsorbed complement component C1q and inhibitors for the
reaction can be readily removed by rinsing. The marker incorporated
in the complex is then quantitatively analysed. For quantitative
analysis, any of the known methods may be used, including visual
measurement, observations through various types of microscopes,
measurement of absorbance of visual and ultraviolet rays,
fluorophotometric measurement and pulse count measurement. In
determination of the marker, other than the direct measurement of
the marker labelled to the combined complex, a known quantity of
the marker is used and the quantity of the marker which has not
been conjugated to the complement component C1q may be determined
to learn indirectly the quantity thereof introduced into the
complex. Anyway, by the quantitative analysis of the marker, the
quantity of the complement-binding antibody coupled to a specific
antigen can be learned.
In the measurement or determination method of the invention,
enzymes are particularly preferred as the signal emitting
substance. Since enzymes act as catalysts, the sensitivities of the
measurement may be freely adjusted by changing the temperature and
time of the reaction.
Various antigens may also been inspected by the measurement method
of the invention. The measurement of a variety of antigens may be
utilized for the detection, identification, quantitative analysis
and inspection of various substances including microorganisms such
as chlamydia and virus, various physiologically active substances
such as interferon and lymphokines, specific antigens for cancers,
specific antigen substances in immunological abnormalities,
allergens in allergic diseases, and medicinal substances such as
hormones; and thus the method may be applied for various purposes,
such as speedy and reliable diagnosis on a variety of diseases,
provision of the standards for judging the effects of curing
actions, inspection for doping, inspection for the determination of
foreign matters in products, and hygienic or sanitary
inspections.
When a specific antigen is measured by the method of the invention,
a substance having affinity with the antigen which substance is
fixed to a solid carrier is reacted with the antigen, and the
complement component C1q labelled with a marker, and optinally with
an antibody, followed by removal of unreacted materials and then
the marker is quantitatively analyesd.
The substances having affinity with the antigen include various
types of substances by which the antigens are readily absorbed,
examples thereof being antibodies, portions of antibodies
containing the sites binding to the antigens [for example, Fab,
F(ab') or F(ab').sub.z ], enzyme substrates and inhibitors, protein
A of staphylococcus, various medicinal substances originated from
organisms, and receptors for virus. A living tissue containing a
substance having affinity with a specific antigen may be used
without being purified. A selected one of these substances having
affinities with antigens is fixed to the solid phase or carrier. By
the use of the fixed phase, rinsing and other operations can be
simplified. Any carriers may be used for this purpose, as far as
the substances having affinities with antigens are not readily
released or removed, and the same carrier materials as has been
described for the method of measuring the complement-binding
antibodies may be used.
A specific antigen to be measured is then added to the fixed
substance having affinity with the antigen so that the antigen is
conjugated with the substance. Any antigens may be measured by the
method of the invention without particular limitation, as fas as
they can be coupled with substances having affinities thereto, such
as the corresponding antibodies. Illustative antigens which may be
measured by the method of the invention include microorganisms such
as viruses and bacteria, products produced by viruses and bacteria,
vital components in animal tissues, physiologically active
substances of plants, and chemicals. Sources for such antigens are
body fluids such as blood, urine, cerebrospinal fluid and saliva,
processed products of meats and plants, and aqueous solutions from
rivers, sewege or waste water.
In the method of measuring an antigen, according to the invention,
the corresponding antibody is added optionally as necessity arises.
When an antibody or a portion of antibody having an
antibody-binding activity and a complement-binding activity is used
as the substance having the affinity with the antigen, it is not
requisite to further add the antibody. However, in case where a
susbstance having no complement-vinding activity is used as the
substance having the affinity with the antigen, it is essential to
add the antibody to be bound to the marker-labelled complement
component C1q. The antibody may be added at any desired time point
after the antigen is added, and may be added simultaneously with
the addition of the complement component C1q or may be added before
or after the addition of the complement component C1q. Of course,
the antibody should be the one which binds uniquely to the specific
antigen to be measured and should be capable of binding with the
complement component C1q. Typical antibodies used commonly are
immunoglobulins contained in animal blood sera, the examples being
IgM, IgG etc. having complement-binding activities. Natural
antibodies present in blood sera may be used, or desired antibodies
may be obtained by administrating or infecting animals with
antigens. In addition to the immunoglobulins purified and separated
from blood sera, inactivated blood sera may be used as the
antibodies without purification.
The condition for the reaction between the antigen, the substance
having affinity with the antigen, and the marker-labelled
complement component C1q and the antibody if it is added is not
ristricted. Only by mixing the materials, the reaction proceeds
spontaneously and quantitatively. While the time and temperature of
the reaction vary depending on the specific kinds of the antigen
and the other reactants, the reaction condition may be set with the
only limitation that the biological activities of the reactants are
preserved.
Since the comlex of the substance having affinity with the antigen,
the antigen and the marker-labelled complement component C1q is
fixed to the solid phase, the unreacted complement component C1q
and inhibitors for the reaction may be easily removed by simple
rinsing operation. The marker of the complex fixed to the solid
phase is then quantitatively analysed. For the quantitative
analysis, similar methods as has been described in determination of
complement-binding antibody may be employed.
While in the above-mentioned methods for measurement a solid phase
or carrier is employed, it is possible to measure without the use
of such a solid phase or carrier. Specifically, after an antigen,
an antibody and a marker-labelled complement component C1q are
reacted, the marker may be measured directly without removing
unreacted materials. In such a case, it is necessary to select a
marker which is highly distinctive and is readily detectable. For
example, if an enzyme such as peroxidase is used as a marker, the
activity or sensitivity of the enzyme can be enhanced as the
concentration of substrates is changed. Therefore, it is preferred
to use enzyme as a marker. As another method in which a solid phase
or carrier is not used, after an antigen, an antibody and a marker
labelled-complement component C1q are reacted, a precipitating
agent is added to precipitate unreacted materials such as unreacted
complement component C1q and other unnecessary proteins followed by
measuting the marker. The examples of the precipitating agent
include materials which lower the solubility of proteins or
agglutinate masses thereof, such as polyethyleneglycol, organic
solvents such as ethanol, inorganic salts such as ammonium sulfate
or sodium sulfate and organic or inorganic acids which change pH
values of solutions such as trichloroacetic acid or hydrochloric
acid. Basic materials may also be used to change the pH value.
Ethylenediamine tetra-acetic acid or ethyleneglycol tetra-acetic
acid may also be used as the precipitating agent since there
compounds capture metal ions in the solutions, thereby lowering the
solubility of proteins or agglutinate masses thereof.
Neutralizing antibodies may also be measured by the method of the
invention. Neutralizing antibodies are antibodies for preventing
infections by microorganisms, such as virus, rickettsia and
chlamydia. Referring to diseases caused by virus, for instance, a
wide variety of viral infectious diseases have been known up to
date, including not a few serious diseases. For instance, if a
pregnant woman is infected with rubella virus, there arises a
danger that a malformed baby is born. Fatal damages are caused by
the infection with rabies virus, Japanese encephalitis virus and
poliovirus, with the nerval cells suffered unrecoverable disorders,
leading to lasting troubles throughout the lifetime. Hepatitis
caused by hepatitis virus is an infectious disease which lasts as a
chronic disease for a very long time, and a portion of the liver is
impaired by liver cirrhosis which might lead to hepatoma.
However, it is extremely hard to inhibit the growth of virus by the
use of a variety of medicines including antibiotics, since a virus
can grow in special living cells, i.e. the susceptible cells,
unlike bacterium and fungi.
Accordingly, it is a more important counter-measure against the
diseases caused by virus to prevent infection by virus or to
protect a person from infection, apart from the curing treatement
of the patients. The judgement on the question whether a person is
susceptible to infection by a specific virus or not may be rendered
by the determination of presence or absence of the neutralizing
antibody to the virus under question and by the measurement of the
titer of the existing neutralizing antibody.
A living body acquires sound immunity after it has been infected
with a specific virus and then recovered from the disease caused
thereby. This means, in fact, that a system for preventing the
living body from re-infection with that virus has been established.
In other words, a living body has been once infected with a
specific virus, the antibody for protecting the body from
re-infection is promoted, the antibody being referred to as
infection preventing antibody. Production and preservation of the
infection preventing antibody are very important factors against
the infection by the virus. After being infected or immunized with
a virus against the attack by the virus (for example inoculated by
vaccine), various antibodies against the structural components of
the virus have been produced in a living body. However, all of
these antibodies produced in the living body are not participated
in the prevention against infection by that virus. Only the
antibody having the function for inhibiting the growth or
propagation of the virus is referred to as neutralizing antibody or
infection preventing antibody. This particular neutralizing
antibody exerts the principal role in prevention of infection. The
present invention provides a method of measuring a variety of
neutralizing antibodies against all viruses, rickettsias and
chlamydias which infect culture cells speedily and quantitatively
on a number of samples.
In the method of measuring a neutralizing antibody, according to
the invention, a liquid containing a known quantity of
microorganism, such as virus, rickettsia or chlamydia, is reacted
with a body fluid to be measured, such as blood, cerebrospinal
fluid, saliva or blood serum. At this reaction step, the
neutralizing antiboy, if present, reacts with the mixed
microorganism. The amount of microorganism reacting with the
neutralizing antibody is increased as the amount of neutralizing
antibody contained in the liquid under measurement increases so
that the amount of the residual microorganism is decreased. Since
the content of microorganism in the measured liquid is known, the
amount of neutralizing antibody contained in the measured liquid
can be calculated from the result of determination of the residual
microorganism.
After the preceding reaction step, the residual microorganism is
inoculated on culture host cells to allow to grow. The cultivation
is stopped after the lapse of pre-set time, whereby fixed cells
containing therein the residual microorganism are obtained. A
marker-labelled complement component C1q and an antibody against
the microorganism are then added to react with the fixed cell to
obtain a modified fixed cell to which the marker-labelled
complement component C1q and the antibody are bound. By the
quantitative analysis of the marker, the amount of residual
microorganism can be learned to find the amount or titer of the
neutralizing antibody.
The residual microorganism is, in general, cultivated initially by
inoculating the microorganism on host cells cultivated through a
monolayer culture on a micro plate to allow the microorganism to be
absorbed by the micro plate, and then allowing it to grow or
propagate on the plate. It is a common practice to inactivate
endogenous enzymes and the microorganism by treating with, for
example, methanol-containing hydrogen peroxide. Antibodies against
the residual microorganism which may be used in the method include
antisera, such as low titer human sera and animal immunoe sera, and
monoclonal antibody.
Furthermore, the materials which may be measured by the method of
the invention, other than those described above, are products
produced or appearing internally of or on the surfaces of cells,
and various microorganisms. The materials belonging to this
category include cell surface antigens produced by cells, such as
asialo GM.sub.1, T antigen and Ly antigen; intracellular enzymes,
such as TdT (terminal deoxynucleotidyl transferase), GTP
(.gamma.-glutamyl transpeptidase) and LDH (lactate dehydrogenase);
secreting substances, such as CEA (carcino embryonic antigen) and
AFP (.alpha.-fetoprotein) and immunoglobulins: and enzymes and
peptide base substances produced by yeasts and bacteria. The cells
referred to above include all kinds of cells including animal
cells, plant cells, heterokaryotes, cells of yeasts, bacteria and
protozoa, and cells subjected to gene engineering. Microorganisms
which may be measured by the method of the invention include all
microorganisms infecting cultivated cells, such as viruses,
rickettsias and chlamydias.
In measurement, one of the aforementioned substances or
microorganisms is cultivated and fixed, or simply fixed, and then
reacted with a complement-binding antibody and a marker-labelled
complement component C1q, followed by determination of the marker,
whereby the substance or microorganism may be quantitatively
analysed.
EXAMPLES OF THE INVENTION
The invention will now be illustratively described with reference
to examples thereof and comparative examples.
EXAMPLE 1
Enzyme-Conjugated Complement Component C1q:
(1) Purification
100 ml of a fresh rabbit blood serum was dialized through 5 l of a
0.026M aqueous solution of ethylene glycol tetra-acetate (pH 7.5)
for 15 to 24 hours, and the formed precipitate was recovered by
centrifugal separation (20,000G, 20 minutes). The recovered
precipitate was dissolved in 20 ml of a 0.75M aqueous solution of
sodium chloride (pH 5.0) containing 0.1M of sodium ethylenediamine
tetra-acetate. After removing the insoluble materials by
centrifugal separation (25,000G, 30 minutes), and then the solution
was dialized through 5 l of 0.063M aqueous solution of sodium
ethylenediamine tetra-acetate (pH 5.0) at 5.degree. C. for 4 hours,
followed by removal of precipitates by centrifugal separation
(20,000G, 20 minutes). About 3 mg of proteins were obtained by the
aforementioned operations, and 95% or more of the thus obtained
proteins was occupied by the complement component C1q. In order to
store the complement component C1q, the proteins were dissolved in
an aqueous solution (pH 7.4) containing 0.05M
tri(hydroxymethyl)aminomethane, 1M sodium chloride, 0.005M sodium
ethylenediamine tetra-acetate and 10% sucrose. The aforementioned
operations sequence may be repeated to further purify the
complement component C1q.
(2) Preparation
30 mg of the thus purified rabbit complement component C1q was
dissolved in 10 ml of an aqueous solution (pH 7.4) containing 0.05M
tris(hydroxymethyl)aminomethane, 1M sodium chloride, 0.005M sodium
ethylenediamine tetra-acetate and 10% sucrose. The solution was
then added with 0.1 ml of a 0.1M dithiothreitol, and allowed to
stand at room temperature for an hour for reaction. The reaction
solution was then passed through a Sephadex G-25 (Trade Name of
Pharmacia Fine Chemicals Co.) column to recover the protein
fraction which was concentrated to have a volume of about 10 ml by
ultrafiltration to obtain 22 mg of a reduced complement component
C1q.
Separately, 20 mg of peroxidase extracted from the horseradish was
dissolved in 6 ml of a phosphate buffer (pH 7.4), and then added
with 4 ml of a dimethylformamide. The solution was further added
with 0.2 ml of a 2% 4-(maleimidemethyl)-cyclohexane-1-carboxylic
acid succineimide ester (hereinafter referred to as CHM) in
dimethylformamide, and then allowed to stand at room temperature
for an hour for reaction. After an hour, the solution containing
the reaction product was passed through a Sephadex G-25 column to
recover 16 mg of a CHM-conjugated peroxidase.
21 mg of the aforementioned reduced complement component C1q and 14
mg of the CHM-conjugated peroxidase were mixed together, and the
mixture was allowed to stand stationarily at 4.degree. C. to 10
.degree. C. for 15 hours and then passed through a Sepharose 6B
(Trade Name of Pharmacia Fine Chemicals Co.) column to recover a
fraction of a molecular weight range of from 400,000 to 800,000 to
obtain 29 mg of a complement component C1q labelled with
peroxidase.
EXAMPLE 2
Enzyme-conjugated Complement Component C1q
1 mg of .beta.-D-galactosidase derived from Escherichia coli was
dissolved in 0.2 ml of a 0.1M phosphate buffer (pH 6.0), and then
reacted with 0.1 mg of N,N'-o-phenylenedimaleimide dissolved in
0.2ml of a phosphate buffer containing 5% dimethylformamide at
30.degree. C. for 25 minutes. The solution containing the reaction
product was passed through a Sephadex G-25 (Trade Name of Pharmacia
Fine Chemicals Co.) column equilibrated with a phosphate buffer
containing 0.2 mg/ml of bovine serum albumins, whereby a 720 .mu.g
of .beta.-D-galactosidase coupled with maleimide.
200 .mu.g of the .beta.-D-galactosidase coupled with maleimide was
dissolved in 0.1 ml of a phosphate buffer containing 1 mM of sodium
ethylenediamine tetra-acetate, and reacted with 2 mg of the recuced
complement component C1q prepared in Example 1 and dissolved in 0.1
ml of a phosphate buffer containing 1 mM of sodium ethylenediamine
tetra-acetate at 4.degree. C. for 48hours. The solution containing
the reaction product was subjected to gel filtration using a
Sepharose 6B (Trade Name of Pharmacia Fine Chemicals Co.) column,
and then processed through the procedures as described in Example 1
to obtain an active fraction, i.e. a fraction containing 1.6 mg of
a complement component C1q combined with
.beta.-D-galactosidase.
EXAMPLE 3
Enzyme-conjugated Complement Component C1q
(1) Preparation
Generally following to the same procedures as in Example 1, except
that a goat serum was used in place of the rabbit serum, a reduced
complement component C1q was prepared, which was then conjugated
with peroxidase to obtain a peroxidase-labelled complement
component C1q.
(2) Test
Using a serum having a CF antibody titer of 16, the reactions of
the thus obtained peroxidase-labelled complement component C1q with
a herpes simplex virus CF antigen and with a normal cell antigen
were inspected by means of the solid phase enzyme immunoassay to
obtain the results as set forth in the following Table 1. In Table
1, the results of this Example are shown together with the results
of the following Comparative Example 1.
COMPARATIVE EXAMPLE 1
Enzyme-conjugated Complement Component C1q Prepared by Conventional
Process and Having the Enzyme Conjugated Generally at Random
(1) Preparation
1.5 mg of horseradish peroxidase was dissolved in 0.2 ml of
distilled water, and added with 60 .mu.l of a 0.1M sodium periodate
solution, followed by agitation at room temperature for 20 minutes.
The solution was dialized through an acetate buffer (pH 4.4)
containing 1M sodium chloride, added with 60 mg of sucrose, and
then added with 1 ml of a carbonate buffer (pH 9.2) containing 3 mg
of purified goat complement component C1q and also containing 1M
sodium chloride and 10% of sucrose. After agitating for 2 hours,
the mixture was further added with 0.1 ml of a 4 mg/ml solution of
sodium borohydride, and then allowed to stand at 4.degree. C. for
additional 2 hours. Thereafter, the admixture was subjected to gel
filtration through a Sephacryl S-300 (Trade Name of Pharmacia Fine
Chemicals Co.) column, and the fraction having both of the
peroxidase activity and the C1q activity was collected.
(2) Test
Using a serum having a CF antibody titer of 16, the reactions of
the thus obtained peroxidase-labelled complement component C1q with
a herpes simplex virus CF antigen and with a normal cell antigen
were inspected by means of the solid phase enzyme immunoassay to
obtain the results as set forth in the following Table 1. In Table
1, the results of this Comparative Example are shown while
comparing with the results of Example 3.
TABLE 1 ______________________________________ Color Development
Color Development of Herpes CF of Normal Antigen Well Antigen Well
______________________________________ Enzyme-Labelled C1q 0.544
0.078 of Example 3 Enzyme Labelled C1q 0.328 0.281 of Comparative
Example 1 ______________________________________
It should be appreciated from the results shown in Table 1 that the
enzyme-labelled complement component C1q prepared by the
conventional process reacts with the normal antigen inselectively
or non-uniquely and and has a low or feeble capability of reacting
with the herpes simplex virus CF antigen selectively or uniquely;
whereas the enzyme-labelled complement component C1q prepared by
the process of the invention is considerably lowered in inselective
or non-unique reaction with the normal antigen to have a
sufficiently high capability of reaction with the herpes simplex
virus CF antigen.
EXAMPLE 4
Toxin-Conjugated Complement Component C1q
(1) Preparation
4mg of purified ricin A chain was disolved in 1.2 ml of a phosphate
buffer (pH 7.0) containing 20% of dimethylformamide, and added with
30 .mu.l of a phosphate buffer containing 3% of
4-(maleimidemethyl)cyclohexane-1-carboxylic acid succineimide ester
(hereinafter referred to as CHM) to react at room temperature for
an hour. Then, the solution containing the reaction product was
passed through a Sephadex G-25 column to obtain 2.3 mg of a
CHM-conjugated ricin which was dissolved in 1 ml of 0.1M phosphate
buffer (pH 6.0) and then added with 2.5 mg of the reduced
complement component C1q prepared by the same process as in Example
3 and dissolved in 0.5 ml of a phosphate buffer to react with the
latter by allowing to stand the mixture at 4.degree. C. for 22
hours. The reaction mixture was subjected to gel filtration using
Sephacryl S-200 (Trade Name of Pharmacia Fine Chemicals Co.) to
obtain 1.9 mg of a ricin-conjugated complement component C1q.
(2) Test
The T cell was refined from the BALB/c mouse spleen cell primed
with DNP-KLH by passing the primed cell through a Nylon wool
column. The T cell fraction was put into a RPMI-1640-10%FCS culture
medium containing anti-mouse Ly-2,3 antiserum and 2 .mu.g/ml of the
aforementioned ricin-conjugated complement component C1q, and then
allowed to stand stationarily at 37.degree. C. for an hour.
Thereafter, the processed cell was rinsed with a Hanks' balanced
salt solution, cultivated in a RPMI-1640-10%FCS culture medium
containing mouse Interleukin 2 for 7 days, and the distribution of
the recovered cell Ly antigen was checked to find that the number
of cells having the Ly-1 antigen on the surfaces thereof were
increased as large as 1.6 times of those of a control which had not
been processed with the anti-mouse Ly-2,3 antiserum and the
ricin-conjugated complement component C1q.
EXAMPLE 5
Dyestuff-Conjugated Complement Component C1q
(1) Preparation
10 mg of purified bovine serum albumin was dissolved in 1 ml of
0.5M carbonate buffer and added with 0.4 mg of fluorescein
isothiocyanate (hereinafter referred to as FITC). After reacting
the mixture for 7 hours, the reaction mixture was subjected to gel
filtration to obtain bovine serum albumin combined with FITC. 8.2
mg of the bovine serum albumin combined with FITC was then
dissolved in 0.4 ml of a 0.1M sodium phosphate buffer (pH 7.0), and
added with 50 .mu.l of a 90 mg/ml solution of CHM in
dimethylformamide for reaction at 30.degree. C. for an hour. After
removing the insoluble materials by centrifugal separation, the
buffer solution was exchanged to 0.1M phosphate buffer (pH 6.0).
0.56 ml of the solution was added with 0.5 ml of a 0.1M phosphate
buffer (pH 6.0) containing 8 mg of the reduced complement component
C1q prepared in Example 1 and 5 mM sodium ethylenediamine
tetra-acetate, and the admixture was maintained at 4.degree. C. for
18 hours. After then, the admixture was filtered through a gel
filter of Sepharose 6B column to obtain 12 ml of an eluate fraction
having a molecular weight ranging within 400,000 to 900,000 and
containing a reaction product between the complement component C1q
and the bovine serum albumin combined with the FITC.
(2) Test
Separately, the spleen cells were removed from the BDF.sub.1 mouse
and passed through a Nylon column to obtain T-cells. The thus
obtained T-cells, a rabbit antiserum against mouse brain associated
T-cell antigen and the eluate fraction prepared through the process
desribed in the preceding paragraph were mixed together to form a
mixture having a concentration of 150 times of the final
concentration. After maintaining the mixture on ice for an hour and
rinsing sufficiently, the cell was floated on the surface of a 50%
glycerin-phosphate buffer solution and observed through a
flourescent microscope. The result was that 93% of the cells
emitted fluorescent light to reveal that almost all of the cells
were the T-cells.
EXAMPLE 6
Complement-Component C1q Conjugated with Donor or Acceptor for
Electron
(1) Preparation
Chlorophyllin a was dissolved in distilled water so that a 1 mg/ml
solution was formed, and the pH value of the solution was adjusted
with hydrochloric acid to pH 7.5, followed by addition of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride and
ethylenediamine so that the resultant reaction mixture contained
0.1M of the former and 0.8M of the latter. The mixture was allowed
to stand for reaction for 120 minutes. The reaction product was
purified by the use of CM-Sephadex (Trade Name of Pharmacia Fine
Chemicals Co.), and 450 .mu.g of aminoethylated chlorophyllin a was
dissolved in 0.5 ml of a 0.1M phosphate buffer (pH 7.0) containing
40% of dimethylformamide. The solution of purified aminoethylated
chlorophyllin a was mixed with 0.5 ml of a 40% dimethylformamide
solution containing 2% of CHM, and maintained at 30.degree. C. for
an hour to react with CHM. The reaction mixture was subjected to
gel filtration using Biogel P-2 (Trade Name of Bio-Rad Laboratories
Inc.) to obtain CHM-chrolophyllin a. 12 ml of the reduced
complement component C1q prepared in accordance with the process as
described in Example 3 and 100 .mu.g of the CHM-chrolophyllin a
were put into 1.5 ml of a 0.1M phosphate buffer (pH 6.0), and
allowed to stand stationarily at 4.degree. C. for 18 hous, followed
by gel filtration at which a fraction having a molecular weight
ranging within 400,000 to 600,000 was picked up, whereby 7.5 mg of
chrolophyllin-labelled complement component C1q was obtained.
(2) Test
Separately, a chemically modified antigen electrode was prepared by
coating an antigen protein against herpes simplex virus on an
SnO.sub.2 Nesa electrode so that the antigen protein was combined
with the electrode through a covalent bond. The electrode was
immersed in a 25mM phosphate buffer (pH 6.95) containing 50 mM
hydroquinone, a 1/50 final concentration of a human blood serum (CF
=32) including an anti-herpes simplex virus, and 10 .mu.g of the
chrolophyllin-labelled complement component C1q, and irradiated by
a white light while maintaining the electrode potential at 0.1
Vs.multidot.SCE, whereupon generation of photocurrents was
observed. The quantum efficiencies of photocurrents were about
9%.
EXAMPLE 7
Magnetizable Substance-Conjugated Complement Component C1q
(1) Preparation
Polystyrene microbeads each having amino groups at the surface
thereof and containing micro grains of magnetite were suspended in
1 ml of 0.1M phosphate buffer (pH 7.0) containing 20%
dimethylformamide, and added with 40 .mu.l of 0.1M phosphate buffer
containing 2.5% CHM, followed by moderate agitation at 30.degree.
C. for 60 minutes for reaction. After rinsing the beads, they were
suspended again in 1 ml of 0.1M phosphate buffer (pH 6.0) and added
with 0.5 ml of another phosphate buffer containing 1.8 mg of the
reduced complement component C1q prepared by the process as
described in Example 3, and then the admixture was allowed to react
at 4.degree. C. for 18 hours under moderate agitation. The beads
were rinsed with a Veronal buffer solution (pH 7.4) containing 0.1%
of gelatin, the solution being referred to as GVB hereinafter, and
then stored in the GVB at 4.degree. C.
(2) Test
Separately, the spleen cells of X5563 tumor-bearing C3H/He mouse
were cultivated on a culture medium containing IL-2, and the once
rinsed cells were again floated on the same culture medium and
mixed with the beads-conjugated complement component C1q prepared
by the process described in the preceding paragraph and an anti
I-Jk antiserum, followed by stationary standing at 37.degree. C.
for 2 hours. After the lapse of the pre-set time, the cells were
collected and then the collected cells were again floated gently,
and an intense magnetical force was applied from the exterior of
the container to capture the cells having the I-Jk antigens at the
surfaces thereof. The cytotoxic activity of the cell left in the
culture medium against the X5563 tumor cell was recognized to be
about 1.4 times higher than that of the cell cultivated for 5 days
in a simple IL-2.
EXAMPLE 8
Measurement of Complement-Binding Antibody
On a 96 well microtiter plate absorbing a complement-fixed antigen
of the herpes simplex virus added, respectively, were 5 .mu.l of
each of sample sera inactivated to have complement-binding titers
of less than 4 and 16, and then 95 .mu.l of the peroxidase-labelled
complement component C1q in Example 1 (diluted to 100 times volume
with a gelatine-Veronal buffer solution) was added, followed by
stationary standing at room temperature for an hour. Thereafter,
each well was rinsed three times with a phosphate buffer containing
0.05% of Tween-20 (a surface active agent produced and sold by
Nakarai Chemicals LTD.), and added with 100 .mu.l of an H.sub.2
O.sub.2 -ABTS solution [2,2'-adino-di-(3-ethyl-benzothiazoline
sulfate) solution containing hydrogen peroxide], followed by
standing at room temperature for an hour to complete the reaction.
After adding with 100 .mu.l of an enzymatic reaction terminating
agent, the light absorbance at 414 nm was measured to find that the
absorbance of the serum having the complement-binding titer of less
than 4 was 0.029 while that of the serum having the
complement-binding titer of 16 was 0.579.
EXAMPLE 9
Measurement of Complement-binding Antibody
Bovine blood serum albumin was dissolved in a saline solution
buffered by phosphate to prepare a solution having a concentration
of 20 .mu.g/ml, which was poured in each well of a 96 well
microtiter plate and then maintained at room temperature for 2
hours to be absorbed by each well. After removing free bovine blood
serum albumin, 50 .mu.l of anti-bovine serum albumin rabbit
antiserum (stepwisely diluted by 800 to 6400 times with a
gelatin-Veronal buffer) and 50 .mu.l of the .beta.-D-galactosidase
-labelled complement component C1q synthesized in Example 2 were
added, and the microtiter plate was allowed to stand at room
temperature for 30 minutes. After rinsing each well, 100 .mu.l of
o-nitrophenyl .beta.-D-galactoside solution (in a phosphate buffer
solution having a pH value of 7.3) was added, followed by standing
at room temperature for 60 minutes, and then 0.1 ml of a 0.1M
sodium carbonate solution to terminate or cease the enzymatic
reaction. The light absorbances of respective wells were measured
at a wavelength of 420 nm to find that the light absorbances were
gradually varied from 0.421 to 0.063 depending on the change in
dosed amounts of antiserum.
EXAMPLE 10
Measurement of Complement-binding Antibody
A purified antigen of herpes simplex virus was absorbed by 6.35 mm
of polystyrne beads which were put into a small test tube, and
added simultaneously with 0.1 ml of a 10 times diluted solution of
a solution of each of inactivated test sera (having the
complement-binding titers of 16 and less than 4) in a
gelatine-Veronal buffer and with 0.1 ml of a solution of the
peroxidase-labelled complement component C1q prepared in Example 1
diluted with the same buffer. The admixture was then allowed to
stand stationarily at room temperature for an hour. After rinsing
the beads, they were transferred to another small test tube, and
added with 0.3 ml of an o-phenylenediamine solution to react at
room temperature for 45 minutes. The reaction was terminated by the
addition of 2 ml of 1 N hydrochloric acid, and the light
absorbances of the samples at the wavelength of 490 nm were
measured. The sample having the complement-binding titer of less
than 4 had a light absorbance of 0.18, whereas the sample having
the complement-binding titer of 16 had a light absorbance of
0.408.
EXAMPLE 11
Measurement of Antigen
A 96 well microtiter absorbing guinea pig anti-herpes simplex virus
antibody (Fab) was supplied with 0.1 ml of uterus cervix swabs of a
patient, and stationarily held at room temperature for 60 minutes.
After rinsing the plate with the PBS (a 0.85% saline-containing
phosphate buffer having a pH value of 7.4) for three times, each
well was added with 0.05ml of either one of the inactivated guinea
pig anti-herpes simplex virus sera (having the complement-binding
antibody titers of 16 to 32) and also with 0.05ml of the
peroxidase-labelled complement component C1q prepared in Example 1.
The plate was held stationarily at room temperature for 60 minutes.
Then, each well was rinsed with PBS containing 0.05% of Tween-20 (a
surface active agent produced and sold under such Trade Name from
Nakarai Chemicals LTD.), and added with 0.01 ml of H.sub.2 O.sub.2
-ABTS [2,2'-adino-di(3-ethyl-benzothiazolin sulfate) containing
hydrogen peroxide]solution to be held at room temperature for an
hour for reaction. Thereafter, 0.05 ml of a 0.05% aqueous sodium
nitride which acted to terminate the enzymatic reaction, and then
the light absorbances of respective sample wells at a wavelength of
414 nm were measured to find that the sample well filled with
uterus cervix swabs of a patient who was negative against the
herpes simplex virus had an absorbance of 0.030 and that the sample
wells filled with uterus cervix swabs of a patient who were
positive against the herpes simplex virus had absorbances of 0.113,
0.300, 0.550 and so on.
EXAMPLE 12
Measurement of Antigen
According to a conventional process, a lymphocyte fraction was
prepared from the mouse spleen cell, followed by rinsing with the
PBS, and then the concentration of the cell was adjusted to
1.times.10.sup.7 /ml. 0.1 ml of the thus prepared lymphocyte
fraction, 0.05 ml of antimouse Thy-1,3 alloserum, and 0.05 ml of
the FITC-labelled complement component C1q of Example 5 were mixed
together and allowed to stand at room temperature for an hour. The
cells were then rinsed thoroughly with the PBS and observed through
a fluorescent microscope. The result revealed that 37% of the cells
were fluorescent.
EXAMPLE 13
Measurement of Antigen
The lymphocytes in a blood of a leukemia patient were suspended in
1 ml of a phosphate buffer to prepare a suspension containing
1.times.10.sup.8 /ml of lymphocytes, and the suspension was
processed by an ultra-sonicator for 2 minutes. The homogenate was
then clarified by centrifugal separation, and the supernatant was
added with 0.5ml of DNA Sepharose to react therewith at 37.degree.
C. for 60 minutes. The DNA Sepharose was bound with DNA related
enzymes, such as DNA polymerase and terminal deoxynucleotidyl
transferase (TdT). Then, 0.1 ml of the peroxidase-labelled
complement component C1q and an inactivated rabbit anti-TdT serum,
followed by reaction at 37.degree. C. for 30 minutes. After rinsing
thoroughly with PBS, the DNA Sepharose was recovered, to which 1 ml
of a solution of H.sub.2 O.sub.2 -ABTS, was added, and the
admixture was reacted at 37.degree. C. for 60 minutes. Then, 1 ml
of a 0.05% aqueous solution of sodium nitride acting as a
terminator for the reaction, and the light absorbance of the
supernatant was measured at a wavelength of 414 nm. It could be
judged that the sample having a light absorbance value of not more
than 0.075 showed that the patient was negative to TdT and that the
sample having a light absorbance value of not less than 0.100
showed that the patient was positive to TdT and suffered from acute
leukemia.
EXAMPLE 14
Measurement of Neutralizing Antibody
Two sample sera having, respectively, neutralizing antibody titers
of 32 and 128 to the HSV (Herpes Simplex Virus) were diluted with a
phosphate buffer to have the volumes four times as large as the
initial volumes, heated at 56.degree. C. for 30 minutes to be
inactivated, and then further diluted with the same buffer to have
egiht times volumes. 0.1 ml for each of the thus inactivated and
diluted sample sera was mixed with 0.7 ml of the same buffer
containing 4.times.10.sup.3 pfu/ml of HSV, and then kept at
37.degree. C. for 60 minutes to proceed the reaction.
Separately, Vero cells had been cultivated through the momolayer
culture on a microplate, onto which a mixture of the serum and the
HSV was added at a content of 50 .mu.l/well, and then the virus was
absorbed by holding the plate in a culture filled with 0.5% carbon
dioxide and maintained at 37.degree. C. for 60 minutes, with the
addition of a maintenance medium followed by cultivation for
additional 24 hours. Then, the cell was fixed by the use of
methanol containing 3% of hydrogen peroxide.
After fixing by the methanol containing 3% of hydrogen peroxide, as
described in the preceding paragraph, 50 .mu.l for each of human
sera having complement-fixing titers against the HSV diluted by 50
times with a gelatine-Veronal buffer (pH 7.4), respectively, of 16
and less than 4 was poured into individual wells, and then each
well was added with 0.2 .mu.g/50 .mu.l/well of the
peroxidase-labelled goat complement component C1q prepared in
Example 3 and dissolved in the same buffer. After allowing to stand
the microplate at room temperature for 2 hours, each well was
rinsed with a phosphate buffer solution containing 0.05% of Tween
20 for three times, and then added with 0.7 ml/well of a H.sub.2
O.sub.2 -ABTS solution to develop coloring of each well which was
subjected to light absorbance determination conducted at a
wavelength of 414 nm. The results are shown in Table 2.
TABLE 2 ______________________________________ Serum for Detection
of Residing Virus OD414 (1)-(2)
______________________________________ Serum for Well (1) Well (2)
Determination of Added with Added with Presence or Absence of
Guinea Pig Normal Neutralizing Antibody Anti-HSV Serum Guinea Pig
Serum Control 0.618 0.095 0.523 (Well Not Added with Sample Serum)
Sample Serum 0.504 0.092 0.412 Having Neutralizing Titer of 32
Sample Serum Having 0.117 0.101 0.016 Neutralizing Titer of 128
______________________________________
EXAMPLE 15
Measurement of Neutralizing Antibody
An anti-HSV positive human blood serum having a neutralizing
antibody titer of 128 and a negative human blood serum were diluted
by four times with a phosphate buffer, and inactivated, and then a
serial dilution series diluted by 4 to 512 times was prepared each
for the both sera on a microtiter plate provided with a number of
wells each having a volume of 0.1 ml/well. Each well was filled
with 0.1ml of a buffer containing 4.times.10.sup.3 pfu/ml of HSV.
The following procedures were the same as in Example 14 to measure
or determine the OD.sub.414. The results are plotted in the graph
illustrated in FIG.1 wherein the abscissa indicates the dilution
rate of each serum and the ordinate indicate the OD.sub.414. As
shown, for the positive serum, the dilution rate giving the value
as large as 1/2 of the maximum OD.sub.414 corresponds to the
neutralizing antibody titer of 128.
EXAMPLE 16
Measurement of Intracellular Substance or Microorganism
A specimen to be inspected was picked up from a defected portions
of HSV infected patient (pendedum or labia), and suspended in 1 ml
of culture medium solution containing an antibiotic. 0.1 ml of the
suspension was inoculated to two wells of a microplate in which
Vero cells had been preliminarily cultivated, and further
cultivated at 37.degree. C. for 22 hours.
After the completion of 22 hour cultivation, the cultivated cells
were fixed with 3% hydrogen peroxide-methanol, and 50 .mu.l of a
human serum diluted by 25 times with a gelatine-Veronal buffer (pH
7.4), the serum having an anti-HSV complement binding titer (CF
titer) of 32 or less than 4, was put into individual wells together
with 50 .mu.l of a solution in the same buffer containing 90 ng of
the peroxidase-labelled complement component C1q prepared in
Example 3. After reacting at room temperature for 2 hours, each
well was rinsed with a phosphate buffer containing 0.05% of Tween
20 for three times, added with 0.1ml/well of a hydrogen
peroxide-ABTS solution (pH 4) followed by standing for an hour to
develop coloring, and then the reaction was terminated by the
addition of 0.1 ml of a 0.01% sodium azide. Thereafter, the light
absorbance of the reaction product in each well was measured. The
well added with the human blood serum having a CF titer of 32 had a
light absorbance of 0.263, whereas the well added with the serum
having a CF titer of less than 4 had a light absorbance of 0.089.
From those result, it could be confirmed that the HSV virion was
present in the specimen inspected.
EXAMPLE 17
Measurement of Intracellular Substance or Microorganism
With the aim to cloning a cell producing carcino embryonic antigen,
the cell T3M-4 producing CEA from pancreas tumor was diluted to the
limit (i.e. to 1 cell/well), and then cultivated on a 96 well
microplate for 16 days. After removing the culture medium solution,
0.1 ml of tripsin-sodium ethylenediamine tetra-acetate was put into
each well to float the cells, and then two plates preliminarily
filled with 0.2 ml/well of a fresh culture medium solution were
replicated so that repricas containing 20 .mu.l/well of floating
cells were prepared. The culture medium solution in one of the
repricas was thrown away, followed by fixation of the cells with
3%H.sub.2 O.sub.2 -methanol, and then added with 0.1 ml of a 1/400
time diluted rabbit anti-CEA antiserum diluted with GVB
(gelatine-Veronal buffer) and 110 ng/0.1ml/well of the
peroxidase-labelled complement component C1q. After reacting at
room temperature for 2 hours and rinsing, coloring of the well was
developed by the addition of a solution of the substrate of ABTS
[diammonium (2,2-azi)-di{3-ethylbenzothiazolin sulfonic acid}]. The
OD.sub.414 of respective wells ranged within 0.127 to 0.386. The
cell in the well showing the maximum OD.sub.414 was picked up from
the other reprica, and subjected to expansion.
EXAMPLE 18
Measurement of Intracellular Substance or Microorganism
100 .mu.g (0.1 ml) of purified .alpha.-fetoprotein and 0.1 ml of
Freund complete adjuvant were mixed together and dosed into the
abdorminal cavity of a 7 week age Blb/C mouse. After 28 days from
the dosage of the aforementioned materials, 100 .mu.g (0.3 ml) of
of AFP (.alpha.-fetoprotein) was additionally dosed, and after 3
days of the dosage of the AFP, the renal cells were picked up and
fused with NS-1 cells. The cells were spread over a 96 well plate
at a concentration or distribution density of 1.times.10.sup.5 /ml.
From the first day to the sixteenth day after fusing, selection by
the HAT medium (hypoxantine-thymidine-aminopterine medium) was
conducted, and the antibody activity of the supernatant of each
cultivated well was inspected on the seventeenth day to reveal that
prodcution of antibody was recognized at a rate of 64/948 wells and
production of anti-AFP antibody was recognized in two wells. The
cells in respective wells were utilized as coated specimens while
being processed by 3%H.sub.2 O.sub.2 -methanol to be fixed, and
then added with 20 .mu.l of a goat anti-mouse IgG (.gamma.-chain
selectivity) diluted by 200 times and also with 18 ng/20 .mu.l of
the peroxidase-labelled complement component C1q, followed by
stational standing for 2 hours. After rinsing thoroughly,
development of coloring of each cell was effected in a
diaminobenzidine solution, and the number of cells producing the
IgG was counted to find that the ratio of positive cells were 62%
and 91%, respectively.
EXAMPLE 19
Measurement without Solid Carrier
0.5 ml of diluted solution of normal guinea pig serum, 0.5 ml of
Herpes Simplex virus and 1 ml of the peroxidase-labelled complement
component C1q solution prepared in Example 1, were charged into a
small test tube, mixed together and reacted at 0.degree. C. for 90
minutes. Alternatively, the same procedures were repeated except in
that 0.5 ml diluted solution of guinea pig serum solution immunized
three times with Herpes Simplex virus was used in place of 0.5 ml
of diluted solution of normal guinea pig serum. Then, 1 ml of each
of polyethyleneglycol solutions was added to each of the reaction
products so that ultimate concentration reached 3%. After each of
the resulting reaction products was allowed to stand at room
temperature, precipitates were removed by centrifugal separation.
Each of these precipitates was again dissolved in 2 ml of a buffer
solution so as to be again processed with polyethyleneglycol.
Finally, a 0.175% H.sub.2 O.sub.2--0.04 % ABTS solution (pH 4.0)
was added to each of the thus processed solutions to develop the
color and the OD 414 value was measured with a spectrophotometer.
It was found that the OD 414 value for the immunized guinea pig
serum was 1.025 while that for the normal serum was 0.092.
EXAMPLE 20
Conjugated Complement COmponents C1q
4 mg of the goat complement component C1q prepared in Example 3 was
dissolved in a tris buffer solution (pH 7.5) and the resulting
solution was reduced with dithiothreitol. The buffer solution was
then replaced by gel filtration by a 0.1M solium phosphate buffer
solution (pH 6.0) containing 5 mM of ethylenediamine tetra-acetate
(EDTA), and the solution was then concentrated to 0.2 mM. The
resulting solution was then admixed with 0.2 ml of the same buffer
solution containing 0.1mg of N,N'-oxydimethylenedimaleimide and the
resulting admixture was maintained at 4.degree. C. for 22 hours.
The unreacted materials were removed by gel filtration to produce
910 .mu.g of conjugated complement components C1q.
EXAMPLE 21
Antibody-conjugated Complement Component C1q
2mg of immunoglobulin G to be specifically bound with sheep blood
cells (anti-SRBC-IgG) was reacted with 150 .mu.g of
4-maleimidemethyl)cyclohexane-1-carboxylic acid succineimide ester
(CHM) in a phosphate buffer solution (pH 7.0). After gel
filtration, the reaction product was concentrated to 0.2 ml. The
resulting product was then reacted with 3 mg of the reduced
complement component C1q dissolved in 0.2 ml of a phosphate buffer
solution containing 2mM of ethylenediamine tetra-acetate (EDTA)(pH
7.0). After gel filtration, 520 .mu.g of the complement component
C1q conjugated with the anti-SRBC-IgG was obtained.
Upon adding the complement component C1q together with the SRBC to
a liquid containing a blood serum antigen (BSA) and an anti-BSA
antibody, agglutination of SRBC occurred to a more or less degree
depending on the amount of the BSA antigen.
Although he present invention has been described with reference to
the specific examples, it should be understood that various
modifications and variations can be easily made by those skilled in
the art without departing from the spirit of the invention.
Accordingly, the foregoing disclosure should be interpreted as
illustrative only and not to be interpreted in a limiting sense.
The present invention is limited only by the scope of the following
claims.
* * * * *